Academic journal article Genetics

The Biologically Relevant Targets and Binding Affinity Requirements for the Function of the Yeast Actin-Binding Protein 1 Src-Homology 3 Domain Vary with Genetic Context

Academic journal article Genetics

The Biologically Relevant Targets and Binding Affinity Requirements for the Function of the Yeast Actin-Binding Protein 1 Src-Homology 3 Domain Vary with Genetic Context

Article excerpt

ABSTRACT

Many protein-protein interaction domains bind to multiple targets. However, little is known about how the interactions of a single domain with many proteins are controlled and modulated under varying cellular conditions. In this study, we investigated the in vivo effects of Abp1p SH3 domain mutants that incrementally reduce target-binding affinity in four different yeast mutant backgrounds in which Abp1p activity is essential for growth. Although the severity of the phenotypic defects observed generally increased as binding affinity was reduced, some genetic backgrounds (prk1Δ and sla1Δ) tolerated large affinity reductions while others (sac6Δ and sla2Δ) were much more sensitive to these reductions. To elucidate the mechanisms behind these observations, we determined that Ark1p is the most important Abp1p SH3 domain interactor in prk1Δ cells, but that interactions with multiple targets, including Ark1p and Scp1p, are required in the sac6Δ background. We establish that the Abp1p SH3 domain makes different, functionally important interactions under different genetic conditions, and these changes in function are reflected by changes in the binding affinity requirement of the domain. These data provide the first evidence of biological relevance for any Abp1p SH3 domain-mediated interaction. We also find that considerable reductions in binding affinity are tolerated by the cell with little effect on growth rate, even when the actin cytoskeletal morphology is significantly perturbed.

TO understand the functioning of cells, it is essential to accurately define the networks of protein- protein interactions that occur within them. To this end, many proteomic studies have aimed to identify large numbers of protein-protein interactions within cells (GAVIN et al. 2002; HO et al. 2002; LI et al. 2004; RUAL et al. 2005). A confounding finding in these studies is that single proteins or protein domains are often seen to interact with many different targets, sometimes numbering in the dozens. At present, there is little known about how these multiple interactions are regulated and whether crucial interaction targets may change under varying environmental conditions or genetic backgrounds. To address these issues, cellular systems involving multiple protein-protein interactions must be analyzed in quantitative detail. In this study, we systematically investigated the in vivo effects of mutations that alter the peptide-binding affinity of the Src-homology 3 (SH3) domain of yeast actin-binding protein 1 (Abp1p).

Abp1p is one of a large roster of proteins involved in actin cytoskeleton regulation and endocytosis that colocalize to cortical actin patches (DRUBIN et al. 1988; MULHOLLAND et al. 1994). Abp1p was originally identi- fied by actin filament affinity chromatography and was first implicated in regulation of the actin cytoskeleton upon observation that overexpression of ABP1 causes defects in actin cytoskeleton organization, bud-site selection, and temperature-sensitive growth (DRUBIN et al. 1988). Although deletion of ABP1 does not cause any readily detectable phenotype, an abp1Δ mutant exhibits synthetic genetic interactions with other conserved actin cytoskeleton regulatory genes: SLA1, SLA2, SAC6, and PRK1 (HOLTZMAN et al. 1993; COPE et al. 1999). Abp1p is a multidomain protein, capable of interacting with other proteins involved in actin cytoskeleton regulation and endocytosis. At its N terminus, it has an actin-depolymerizing factor/cofilin homology domain that is required for actin filament binding and activation of the Arp2/3 complex in vitro, followed by two acidic motifs that are also involved in Arp2/3 activation (DRUBIN 1990; COPE et al. 1999; GOODE et al. 2001). Abp1p also contains a proline-rich region and a Cterminal SH3 domain, which are required for mediating protein-protein interactions with other cortical actin patch proteins (LILA and DRUBIN 1997; FAZI et al. 2002; WARREN et al. 2002; STEFAN et al. …

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